Luminescent dyes are widely used markers for many applications in fields of high impact such as environmental and food analysis, security, and medical diagnostics. Fluorescence measurements are usually very sensitive, low cost, easily performed, and versatile, offering submicron visualisation and sub millisecond temporal resolution (L. Prodi, New J. of Chem. 2005, 29, 20-31).
In particular, since an early diagnosis is strictly connected to the success of the therapy and to the quality of life of the patient, medical diagnostics requires luminescent labels and sensors endowed with specific (photo)chemical and photophysical properties, including water solubility, photostability, very low toxicity and high brightness (L. Prodi, New J. of Chem. 2005, 29, 20-31; Wolfbeis, O. S. Analytical Chemistry 2006, 78, 3859).
The versatility of photoluminescence spectroscopy originates also from the wide number of parameters that can be tuned in order to optimize the convenient signal. Even very complex analytical problems can be indeed overcome by controlling the excitation and emission wavelengths, the time window of signal collection, and the polarization of the excitation beam or of the emitted light.
A valuable fluorescence-based label must present different features (O. S. Wolfbeis, Anal. Chem. 2006, 78, 3859-3873). As all bio-labels, it is requested to present reactive groups for the covalent coupling to biomolecules, to be water soluble and non toxic. As far as the fluorescent properties are concerned, the first one relies on the fact that the fluorescent unit should give the highest possible luminescence signal. Reminding that photoluminescence is a two-steps process, since it involves the formation of the excited state through the absorption of a photon and its consequent radiative deactivation, the signal intensity is directly related to the efficiency of both processes through the molar absorption coefficient (ε) and the luminescence quantum yield (Φ). The luminescence intensity in fact, in very diluted solutions is directly proportional to the product ε×Φ, that is defined as the brightness of the dye (L. Prodi, ibid.). Photostability is also particularly important, especially in imaging applications. Furthermore, autofluorescence and light scattering, sources of relevant interferences particularly when biological samples are involved, have to be avoided in order to increase the signal-to-noise ratio. This can be done typically using three distinct approaches. The first one is based on the development and use of red and Near Infra-Red (NIR) dyes, that show absorption and luminescence bands in the 700-900 nm region. These dyes offer minimal background as a result of reduced scattering (due to the inverse 4th power dependence on the wavelength) and of the absence of natural fluorescence of biomolecules in this spectral range. The second approach is based on the use of phosphorescent dyes with long lifetimes at room temperature. In this case, the background light is excluded by the use of time-resolved spectroscopy, since the scattered light and the fluorescence from natural fluorophores decay much faster than phosphorescence, and can be therefore eliminated by the measuring arrangement. Finally, a large Stokes-shift can also be of value, since it helps to reduce the interferences from the Rayleigh-Thyndall and Raman bands.
Because of the wide application of luminescence spectroscopy, huge research efforts have been spent to optimize the design of fluorescent labels, also taking profit of the advances in the nanotechnology arena (Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013; Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem. Int. Edit. 2009, 48, 872; Shi, D. L. Adv. Funct. Mater. 2009, 19, 3356; Gunasekera, U. A.; Pankhurst, Q. A.; Douek, M. Targeted Oncology 2009, 4, 169; Strassert, C. A.; Otter, M.; Albuquerque, R. Q.; Hone, A.; Vida, Y.; Maier, B.; De Cola, L. Angew Chem Int Edit 2009, 48, 7928, Doshi, N.; Mitragotri, S. Adv. Funct. Mater. 2009, 19, 3843; Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nature Materials 2005, 4, 435). Among all the different possibilities offered by this research field, silica based luminescent nanoparticles (also known as Dye Doped Silica Nanoparticles—DDSNs) can offer intriguing solutions for important analytical problems, particularly those related to medical diagnostics and imaging (D. Shi, Adv. Funct. Mat. 2009, 19, 3356-3373), and for the development of nanotheranostic devices (Shi, D. L. Adv. Funct. Mater. 2009, 19, 3356; Gunasekera, U. A.; Pankhurst, Q. A.; Douek, M. Targeted Oncology 2009, 4, 169; Yong, K. T.; Roy, I.; Swihart, M. T.; Prasad, P. N. Journal of Materials Chemistry 2009, 19, 4655; Kim, D. K.; Dobson, J. Journal of Materials Chemistry 2009, 19, 6294; Liu, Y. Y.; Miyoshi, H.; Nakamura, M. International Journal of Cancer 2007, 120, 2527; Liu, Y.; Lou, C.; Yang, H.; Shi, M.; Miyoshi, H. Curr. Cancer Drug Targets 2011, 11, 156). Silica, in fact, does not present intrinsic toxicity, although deeper investigations are underway to completely rule out possible hazards related to the tiny dimensions of nanoparticles (Gunasekera, U. A.; Pankhurst, Q. A.; Douek, M. Targeted Oncology 2009, 4, 169; Wang, L.; Wang, K. M.; Santra, S.; Zhao, X. J.; Hilliard, L. R.; Smith, J. E.; Wu, J. R.; Tan, W. H. Analytical Chemistry 2006, 78, 646; Yong, K. T.; Roy, I.; Swihart, M. T.; Prasad, P. N. Journal of Materials Chemistry 2009, 19, 4655; Burns, A. A.; Vider, J.; Ow, H.; Herz, E.; Penate-Medina, O.; Baumgart, M.; Larson, S. M.; Wiesner, U.; Bradbury, M. Nano Letters 2009, 9, 442). Moreover, their quite simple and affordable synthesis can easily lead to water-soluble systems ready for bio-conjugation. In addition, each DDSN can contain many fluorophores and reach a molar absorption coefficient that easily overcomes 106 M−1 cm−1. The silica matrix can also protect the dyes segregated inside the nanoparticle from external chemicals, thus increasing their (photo)stability and, in many cases, their luminescence quantum yield, so that DDSNs generally show impressively high brightness.
Besides brightness, however, DDSNs can present also the other features discussed above.
For example they can be easily engineered to present a large Stokes-shift and, in more demanding conditions, to present suitable properties for barcoding and multiplexing analysis. The simplest strategy proposed so far for obtaining a large separation between excitation and emission wavelength is the one used by Wiesner and coworkers (E. Herz, A. Burns, D. Bonner, U. Wiesner, Macromol. Rapid Commun. 2009, 30, 1907-1910), who synthesised DDSNs containing commercial fluorophores characterized by an intrinsic large Stokes shift (typically having the lowest excited state with a charge transfer character) and derivatized with an alkoxysilane group. This approach is straightforward but may be limited by the relatively small amount of dyes with this property.
Another, most interesting and fruitful approach is to exploit efficient energy transfer processes between two or more species, metal complexes or organic dyes, that are confined inside the silica nanoparticle. Zhao and co-workers (C. Wu, J. Hong, X. Guo, C. Huang, J. Lai, J. Zheng, J. Chen, X. Mu, Y. Zhao, Chem. Commun. 2008, 750-752) developed a silica based system doped with Ru(II) and Tb(III) complexes, while Konovalov and co-workers (S. V. Fedorenko, O. D. Bochkova, A. R. Mustafina, V. A. Burilov, M. K. Kadirov, C. V. Holin, I. R. Nizameev, V. V. Skripacheva, A. Yu. Menshikova, I. S. Antipin, A. I. Konovalov, J. Phys. Chem. C 2010, 114, 6350-6355) proposed an analogous system, but even more red shifted, based on species containing Ru(II) and Yb(III).
This strategy can also allow to obtain a set of nanoparticles presenting emissions of different colours, but that can be efficiently excited at the same wavelength (L. Wang, W. H. Tan, Nano Lett. 2006, 6, 84-88; L. Wang, C. Y. Yang, W. H. Tan, Nano Lett. 2005, 5, 37-43; X. L. Chen, M. C. Estevez, Z. Zhu, Y. F. Huang, Y. Chen, L. Wang, W. H. Tan, Anal. Chem. 2009, 81, 7009-7014), a feature that is otherwise achievable only using Quantum Dots (QDs). It is important to note that, depending on the number and nature of the dyes and the efficiency of the energy transfer processes among them, two different applications can be figured out.
The first one is based on the development of barcoding NPs. In this case, a family of different nanoparticles is prepared using a set of n dyes, each one giving distinguishable luminescence band, as doping material. Each kind of nanoparticle is characterized by a different concentration of the various dyes inside the silica matrix. If the dyes are suitably chosen in order to have a partial but not complete energy transfer, all nanoparticles can exhibit a multiband emission under one wavelength excitation, and they can be distinguished by a signature constituted by different intensities at the n bands (colours) of the different n dyes. Using NPs doped with four dyes presenting 5 different intensities at each of the four emission bands, 1024 (44) different nanoparticles can be envisaged. This approach is of help whenever a single nanoparticle can be addressed, for example in fluorescence microscopy or flow-cytometry: if each kind of nanoparticle is derivatized in order to recognize a different biomolecules or biostructure (cell), the fluorescence signature of the NPs indicates unambiguously the nature of the analyte under investigation, allowing extensive multiplexing. It is important to remind that the ability to simultaneously measure the amount of many analytes in a single assay, is becoming more and more important in medical diagnostics and imaging (Wolfbeis, O. S. Analytical Chemistry 2006, 78, 3859; Yao, G.; Wang, L.; Wu, Y. R.; Smith, J.; Xu, J. S.; Zhao, W. J.; Lee, E. J.; Tan, W. H. Analytical and Bioanalytical Chemistry 2006, 385, 518; Sukhanova, A.; Nabiev, I. Critical Reviews in Oncology Hematology 2008, 68, 39). Many examples are reported of this approach also for silica nanoparticles.
The second approach is possible only when a (almost) complete energy transfer occurs between the different dyes present inside the nanoparticle. If a set of four different dyes, A, B, C, and D (in order of increasing wavelength) are used, a set of four nanoparticles can be obtained containing (i) A, (ii) A and B, (iii) A, B, and C and (iv) A, B, C, and D. All nanoparticles could be excited at the absorption of A (single wavelength excitation) but, in this case, only the longest-wavelength dye can exhibit significant fluorescence even at short-wavelength excitation (Wang, L.; Tan, W. H. Nano Letters 2006, 6, 84). Although in this case the number of possible analytes that can be investigated at the same time is significantly lower, it is possible to distinguish the different signals also without the need to separate the different nanoparticles, a feature that is of interest, for example, in many DNA analysis, cytofluorimetry and histochemistry. In this context, DDSNs can be a valuable alternative to the commercial Tandem Dyes, that are a combination of two fluorochromes, an energy donor, such as phycoerythrin, and an energy acceptor (typically Cyanine 5 or 7) (Roederer, M.; Kantor, A. B.; Parks, D. R.; Herzenberg, L. A. Cytometry 1996, 24, 191).
Tandem dyes, while offering high brightness and large Stokes-shifts, presents many drawbacks, such as instability and variability (Hulspas, R.; Dombkowski, D.; Preffer, F.; Douglas, D.; Kildew-Shah, B.; Gilbert, J. Cytom. Part A 2009, 75A, 966).
There is also the need to provide highly reproducible and stable labels based on silica nanoparticles doped with (at least) two different dyes in which a very efficient energy transfer process between them could ensure an almost quantitative quenching of the donor and sensitization of the acceptor.
It is also to note that a proper design of efficient intra-particle energy transfer could yield DDSNs suitable to perform other highly valuable functions such as light harvesting, signal processing and energy conversion (Bonacchi, S.; Genovese, D.; Juris, R.; Montalti, M.; Prodi, L.; Rampazzo, E.; Zaccheroni, N. Angew. Chem. Int. Ed. 2011, 50, 4056). To our knowledge, this kind of approach is still unexplored in the field of silica nanoparticles.
Nanoparticles are used in the bio-analytical field, in particular for the detection, labelling and imaging of biomolecules and also as therapeutics, especially as drug carriers (see for example Q. Huo, J. Liu, L. Q. Wang, Y. Jiang, T. N. Lambert, E. Fang, J. Am. Chem. Soc. 2006, 128, 6447-6453).
Tan and co-workers (L. Wang, W. Tan, Nano Lett. 2006, 6, 84-88 and in WO2007044711) disclose dual- and triple-dye nanoparticles for detection of microorganisms and biological material. According to these references, one other potential advantage of Fluorescence Energy Transfer Process (FRET) NPs is that by optimizing the amount of dye molecules in an NP, the emission spectrum can be tuned so that only the longest-wavelength dye will exhibit significant fluorescence at a short-wavelength excitation. This feature will overcome the challenge of the small Stokes shift of many organic dyes, enabling the NPs to be detected in samples with significant Rayleigh/Raman scattering or with endogenous fluorescece. However there are no indications on how to find a solution to this problem or to provide any improvement. Moreover, energy transfer efficiencies are lower and the “noise” of the channels different from the main one is high. By this reason, the authors suggest application also in bar-coding, where for each channel (colour) there are different intensities (for example 4, which with 5 channels give 54 (1024) kinds of different nanoparticles, each one can be associated with a given biomarker). This kind of application is unsuitable for the purposes of the present invention.
L. Wang, W. Zhao, W. Tan, Nano Res. 2008, 1, 99-115 review the use of bioconjugated silica nanoparticle in therapy and diagnostics. In this review, the authors propose two- and three-dye doped silica nanoparticle for multiplexed bacteria detection. A three dye nanoparticle is described, the three dyes were chosen to allow efficient fluorescence energy transfer and are fluorescein isothiocyanate (FITC), rhodamine 6G (R6G) and 6-carboxyl-X-rhodamine (ROX) because of their effective spectral overlapping.
A number of diagnostic techniques using Energy Transfer Process (or Fluorescence Energy Transfer Process—FRET) require high brightness associated with a large Stokes shift.
This implies the highest possible efficiency for energy transfer process, the possibility of single wavelength excitation and large Stokes shift.
However, there is still the problem of parasite self-quenching of the donor dye.
Furthermore, in the search of the highest brightness possible, self-quenching processes have to be minimized, since they reduce the average fluorescence quantum yield limiting the validity of the direct approach to increasing the molecule brightness by increasing the extent of labelling (Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 3rd Ed. ed.; Springer: New York, 2006; Montalti, M.; Prodi, L.; Zaccheroni, N.; Zattoni, A.; Reschiglian, P.; Falini, G. Langmuir 2004, 20, 2989).
In this context, it has to be underlined that also inside DDSNs self-quenching processes can occur, although the observed decrease in the quantum yield is often more than counterbalanced by the increase of the absorption due to the high number of dyes included in the core of the nanoparticles. Efficient energy transfer inside DDSNs can help in this direction.
In particular, coumarinic dyes, or xanthene dyes such as fluorescein, are good donors in energy transfer process, but suffer of parasite self-quenching when loaded in nanoparticles in particular concentrations (see for example: M. Montalti, L Prodi, N. Zaccheroni, A. Zattoni, P. Reschiglian, G. Falini, Langmuir, 2008, 20, 2989-2991).
As far as known to the present inventors, the solution to the problem of self-quenching and at the same time assuring an efficient energy transfer process and a large Stokes shift in multiplexing analytical and diagnostic techniques has not yet been reported in the literature.